Phospholipids such as phosphatidylserines (PSs) and phosphatidylglycerols (PGs) each have their useful physiological or biological functions and specific physical properties and are used in, for example, pharmaceutical preparations, food materials, and emulsifying agents. For example, phosphatidylserines are promising as drugs for prophylaxis and/or therapy of senile dementia and dysmnesia (memory disorder), phosphatidylglycerols are promising as emulsifying agents, and phosphatidylascorbic acids are promising as emulsifying agents and lipoperoxides inhibitors.
These phospholipids have been conventionally produced by chemical synthesis or by transphosphatidylation using phospholipase D. Among these production methods, enzymatic methods can relatively easily produce phospholipids at relatively low cost and are widely used.
Methods for producing objective phospholipids by transphosphatidylation (phospholipid base exchange reactions) have been known from a long time ago (Yang, S. F. et al., J. Biol. Chem., 242, p. 477, 1967). For example, Kokusho et al. disclose that a reaction product containing phosphatidylserine is obtained by a biphasic reaction in which phospholipase D is allowed to act upon a mixture of a solution of egg-yolk phosphatidylcholine in isopropyl ether with a L-serine aqueous solution containing calcium chloride (Agric. Biol. Chem., 51, p. 2515, 1987). It is generally believed that a reaction system in such a biphasic reaction comprises two phases of an oil phase containing a material phospholipid and an aqueous phase containing an acceptor and that transphosphatidylation occurs at the interface between the two phases.
Japanese Patent No. 2,942,302 describes a homogenous reaction in which a phospholipid preparation containing about 85% of phosphatidylcholine prepared by fractionating soybean lecithin is dissolved in ethyl acetate, the resulting solution is mixed with an ascorbic acid aqueous solution to yield a mixture, and the mixture is allowed to react with phospholipase D to thereby yield a reaction product containing phosphatidyl ascorbate.
However, the biphasic reaction must be carried out in the presence of solvents (an organic solvent and water) five times or more (volume/weight) as much as the phospholipid and thereby must use a reactor having a volume capacity six times as much as the amount of the phospholipid. In addition, calcium added to accelerate the reaction rapidly forms a salt with the phospholipid. The formed calcium salt is belonging to the category of chemically synthesized substances in Japan and Europe, and the product is thereby difficult to use in food.
In the homogenous reaction (monophasic reaction), the reaction system contains large amounts of water and yields a phosphatidic acid, as a by-product, due to hydrolytic activity of phospholipase D during a continuous reaction, thus the separation and purification of the objective phospholipid becoming difficult. In addition, the proportion of the acceptor to the phospholipids is limited in the homogenous reaction, and thereby the production amount of the objective reaction product (phospholipid) is limited.
A patent granted to Fujita et al. (JP-B-7-016426) describes that phosphatidylserine, phosphatidylglycerol, and others are produced by a reverse micelle reaction in which an aqueous phase containing calcium chloride, a hydroxyl-containing acceptor, and phospholipase D and being encapsulated in a reversed micelle is allowed to react with a solution of a raw material phospholipid in an organic solvent (diisopropyl ether, isooctane, cyclohexane, benzene, chloroform-isooctane, n-hexane, or dichloromethane-isooctane).
In the Japanese Patent Publication, Fujita et al. report that the reverse micelle reaction requires only a small amount of water and thereby suppresses the formation of phosphatidic acids, the problem of the above method. However, the method in question insufficiently yields the objective phospholipid in a yield of at most about 20%, requires complicated operations such as ultrasonic treatment and thus invites problems in operability and cost. The method also requires the organic solvent 10 times (volume/weight) as much as the phospholipid and must use a reactor having a capacity many times as much as the amount of the phospholipid.
These conventional transphosphatidylation reactions must be carried out in a reaction system containing an organic solvent. However, when product phospholipids are used in, for example, food and pharmaceutical preparations, the organic solvent must not remain in the products and must be completely removed. Accordingly, their production process steps require facilities for the removal of the organic solvent, thus inviting disadvantages in; for example, operability and cost. In particular, such organic solvents cannot be substantially used in the reactions based on the food sanitation law when the products are used in the production of food.
Demands have therefore been made on methods for producing phospholipids without using organic solvents and/or calcium salts. However, one skilled in the art generally believes that a reaction does not smoothly proceed without using organic solvents and thereby the yield of the objective phospholipid and operability should decrease, since the material phospholipids such as phosphatidylcholines are oil-soluble.